Small Column Research Articles

Online restricted access molecularly imprinted solid phase extraction coupled with electrospray ionization-tandem mass spectrometry for determination of mebendazole and albendazole in milk samples

Multifunctional materials, such as restricted access molecularly imprinted polymers covered with bovine serum albumin (RAMIP-BSA), are effective alternatives for sample preparation techniques. This material selectively adsorbs analytes while excluding macromolecules, enhancing the analysis's efficiency. Among analytical techniques, ESI-MS/MS (Electrospray Ionization-Tandem Mass Spectrometry) has successfully identified and quantified various molecules, including trace-level drugs. Therefore, we proposed, for the first time, an integrated online extraction/analysis system that combines the benefits of RAMIP-BSA and ESI-MS/MS for analyzing mebendazole (MBZ) and albendazole (ABZ) in milk samples without the need for chromatographic separation. Initially, a RAMIP selective for MBZ was synthesized using the bulk method with methacrylic acid and glycidyl methacrylate. Then, the polymer was covered with bovine serum albumin. Subsequently, this adsorbent was packed in a small column and coupled with an ESI-MS/MS instrument in an online configuration. Milli-Q water was used as the loading and reconditioning mobile phases, and a solution of formic acid in methanol (1:100 v/v) was employed as the elution phase. The system enabled simultaneous extraction and determination of MBZ and ABZ in milk samples. The method exhibited linearity between 15.0 and 125.0 μg L−1 for MBZ and 10.0 and 125.0 μg L−1 for ABZ (with a correlation coefficient exceeding 0.99). The limits of quantification were 15.0 and 10.0 μg L−1 for MBZ and ABZ, respectively. Good precision and accuracy were achieved. The developed method was used to analyze MBZ and ABZ in real milk samples and proved to be a viable alternative to conventional sample preparation and chromatographic techniques.

Modelling Approach for the Continuous Biocatalytic Synthesis of N-Acetylneuraminic Acid in Packed Bed Reactors

Continuous flow technologies have become increasingly important for biocatalytic processes. In this study, we present the application and modelling of covalently immobilised N-acetylglucosamine 2-epimerase and N-acetylneuraminic acid lyase in packed bed reactors for the synthesis of N-acetylneuraminic acid. The immobilised enzymes were stable under continuous flow process conditions with half-life times of >28 d (epimerase immobilised on hexamethylamino methacrylate HA403/M) or 58 d (lyase immobilised on dimenthylamino methacrylate ECR8309M), suitable for continuous flow applications. Kinetic studies revealed Michaelis–Menten kinetic behaviour for both enzymes. The kinetic parameters and the inhibitions were analysed under continuous flow conditions and were integrated into a process model using Python. The model was validated by varying flow rates, the mass of immobilised enzymes and the reactor dimensions and shows a low error compared to the measured data. An error accuracy of 6% (epimerase) or 9% (lyase) was achieved. The product concentrations of the enzyme cascade at the end of the packed bed reactor can be predicted with an accuracy of 9% for the calculation of a large column (84.5 mL) or of 24% if several small columns (2.5 mL, 0.8 mL) are connected in series. The developed model has proved to be valid and will be used to optimise the process with respect to substrate concentrations, reactor dimensions and flow rate.

Development of a selective sequential process for H2S enrichment and CO2 capture in aqueous MDEA solutions

The typical acid gas removal process can simultaneously remove acid gases from the raw gas using an aqueous MDEA solution. But it cannot further separate and capture hydrogen sulfide and carbon dioxide, and increases the burden on subsequent processing. Recovering carbon dioxide from that not only increases the H2S content in the acid gas, but also achieves the CO2 capture. To realize these, acid gas enrichment and carbon dioxide capture are integrated into the typical acid gas removal process. In this study, a novel selective sequential process in aqueous MDEA solutions is proposed for H2S enrichment and CO2 capture. And an optimization framework is proposed to optimize the parameters of the whole process, mainly to improve the efficiency of the column equipment. It is found that small diameter column is suitable for enhancing selectivity and large diameter column is suitable for maximizing productive capacity. To demonstrate the performance of the proposed process, the evaluation results, carbon sulfur ratio (3.81), return on investment (6.26) and carbon dioxide capture ratio (−0.63) are obtained from technical, economic and environmental evaluations. In comparison with the typical process, the novel process can achieve H2S enrichment and CO2 capture while remaining economically viable. This study presents a method for collaborative removal of H2S and CO2 in acid gas removal process. It is advantageous to reduce carbon dioxide emissions.

Water Column Nitrogen Removal During Storms in a Low‐Order Watershed

AbstractWater column removal in streams is a nitrogen (N) cycling pathway that has been historically overlooked. Studies filling this knowledge gap have focused on the role of water column N removal in mid‐to‐large‐order rivers with consistently high suspended sediment concentrations. However, smaller streams may provide comparable suspended sediment concentrations during and after storm events, creating favorable conditions for water column N removal. To assess the presence, magnitude, and control of water column N removal during storms in low‐order watersheds, we measured water column denitrification and heterotrophic assimilatory N uptake rates at three locations in a Mid‐Atlantic watershed during five storm events of different magnitude, sediment loads, and nutrient availability. We found large variations in water column denitrification (0–5.56 mg N g−1 d−1) and assimilatory uptake (0.003–1.67 mg N g−1 d−1). Higher rates of N removal occurred during flow recession, with a correlation between suspended sediment organic matter content and denitrification. On average, denitrification rates in the water column were higher when flashy responses to storm events occurred. In contrast to denitrification, water column N removal rates (as both denitrification and heterotrophic assimilation) during storm events were comparable to those measured at baseflow in larger rivers. However, water column denitrification could only account for less than 10% of potential reach‐scale N removal during most of the storm events. Our findings provide insight into the ecological relevance of small stream water columns and suggest that more research is needed to understand the magnitude of stream water column processing on watershed‐scale N removal.

Solute transport characteristics of columnar volumetric contraction networks with mega column structure and aperture variability

Numerical simulations explore for the first time the role of mega columns and aperture variability on particle transport through mature volumetric contraction networks as informed by a unique synthesis of network propagation and maturity. Columnar fracture patterns are generated by updating a series of Voronoi centers to the midpoint of a generated polygon over many iterations, creating 250 network realizations. A DFN simulator solves for fluid flow and tracks conservative particle trajectories within each network. Dominant fracture attributes impacting fluid flow and solute transport in volumetric contraction networks are fracture orientation, density, and aperture/transmissivity. Ensemble plume snapshots generated by networks with equal fracture transmissivity define a baseline-level of dispersion that is solely attributed to network structure and connectivity. Longitudinal and transverse dispersion increase and the center of plume mass becomes delayed relative to the baseline case when fracture transmissivity is varied according to a lognormal distribution. The incorporation of highly-transmissive, large-aperture mega column fractures leads to plume snapshots with a more pronounced leading edge and an order of magnitude faster average breakthrough times. The breakthrough curves contain three peaks reflecting contrasting transport pathways in which particles are: (i) initially placed in mega column fractures and remain in these features until exiting the model domain, (ii) initially placed into small column fractures, incur additional time to migrate and enter a mega column fracture, and remain within those mega columns, and (iii) initially placed in small column fractures and remain in these fractures. Incorporating variability in fracture transmissivity for both small column and mega column fractures disrupts the binary distinction between small column and mega column fracture velocities and leads to dispersed breakthroughs over long time scales with a single peak. These results demonstrate that preferential flow paths emerge in volumetric contraction networks due to contracts in fracture transmissivity, not fracture connectivity as observed in tectonic networks.

Ultrasonic preparation of fluorinated functionalised magnetic covalent organic frameworks for adsorption and removal of tetrabromobisphenol A

Tetrabromobisphenol A (TBBPA) was a widely used brominated flame retardant, and had attracted widespread attention from researchers due to its potential adverse effects on ecosystems and human health. Therefore, to eliminate the negative effects of TBBPA, methods for the adsorption and removal of TBBPA are urgently needed. In this work, the fluorine-functionalised magnetic covalent organic frameworks (Fe3O4@TFAPT-TFPA@COF) were first prepared through a simple and rapid ultrasound method to efficiently adsorpt and remove TBBPA. Firstly, a pre-modification strategy was used to modify fluorine functional groups onto amine monomers via superacid catalyzed trimerization. Then, amine and aldehyde monomers undergo Schiff base reaction smoothly to form a COF layer coated on the surface of Fe3O4 nanoparticles. The characterisation results indicated that the prepared Fe3O4@TFAPT-TFPA@COF had a high surface area, porosity, crystallinity and abundant F-containing binding sites. Additionally, the adsorption process of prepared Fe3O4@TFAPT-TFPA@COF complied with pseudo-second-order kinetics and the Langmuir adsorption model. It had a large adsorption capacity (107.5 mg g−1), could effectively remove tetrabromobisphenol A within 4 minutes and had excellent regeneration ability within eighth repetitions. Finally, tetrabromobisphenol A was efficiently removed by a small separation column loaded with Fe3O4@TFAPT-TFPA@COF, achieving levels below the detection limit (< 0.05 mg L−1) and providing practical application value for the real-time removal of TBBPA. The main mechanisms promoting the excellent adsorption performance of TBBPA were through halogen bonds, electrostatic interactions, π-π stacking, and hydrogen bonding interactions, providing potential reference value for the adsorption ability and mechanism of TBBPA in other applications.

Axial compression performance of rubberized concrete-filled steel tubular stub columns after fire exposure: Experimental investigation and calculation models

Rubberized concrete (RuC) has emerged as a promising eco-friendly solution for structural elements, offering improved energy dissipation, damping characteristics, and ductility. Incorporating rubber particles into concrete-filled steel tubular (CFST) columns presents an environmentally conscious alternative for various structural applications. However, understanding its performance post-fire is crucial for safe design application, an area currently lacking comprehensive research. This study investigates the structural behavior of sixteen axially loaded circular RuCFST columns after exposure to fire. The columns, with diameters of 219 mm and 377 mm, are filled with normal concrete and RuC, containing 5 %, 15 %, and 30 % replacement of natural fine and coarse aggregate with rubber particles. Following ISO-834 fire standards, the specimens undergo elevated temperatures and subsequent application of compressive axial loads to determine their post-fire behavior. Analysis includes examination of failure modes, temperature-time relationships, axial load-deformation curves, and strain and stress development in the columns. Microstructural analysis using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) is conducted to study internal morphology before and after exposure to high temperatures. Results indicate non-uniform and nonlinear distributions of maximum temperatures across cross-sections, with historical temperature affecting residual capacity more significantly with increased rubber content and decreased column size. Load-bearing capacities decrease by up to 30 % and 22 % for small and large RuCFST columns, respectively, after exposure to fire. Residual axial stiffness also declines, with reductions of nearly 55 % and 42 % for small and large columns, respectively. In contrast, ductility improves in fire-exposed specimens, with columns having a 30 % rubber replacement ratio showing the highest enhancement. A novel method for calculating post-fire residual load capacity based on the average historical maximum temperature is introduced, demonstrating high accuracy and offering a valuable tool for assessing and reinforcing RuCFST columns after fire exposure.

A fast and sensitive size-exclusion chromatography method for plasma extracellular vesicle proteomic analysis.

Extracellular vesicles (EVs) carry diverse biomolecules derived from their parental cells, making their components excellent biomarker candidates. However, purifying EVs is a major hurdle in biomarker discovery since current methods require large amounts of samples, are time-consuming and typically have poor reproducibility. Here we describe a simple, fast, and sensitive EV fractionation method using size exclusion chromatography (SEC) on a fast protein liquid chromatography (FPLC) system. Our method uses a Superose 6 Increase 5/150, which has a bed volume of 2.9mL. The FPLC system and small column size enable reproducible separation of only 50µL of human plasma in 15min.To demonstrate the utility of our method, we used longitudinal samples from a group of individuals who underwent intense exercise. A total of 838 proteins were identified, of which, 261 were previously characterized as EV proteins, including classical markers, such as cluster of differentiation (CD)9 and CD81. Quantitative analysis showed low technical variability with correlation coefficients greater than 0.9 between replicates. The analysis captured differences in relevant EV proteins involved in response to physical activity.Our method enables fast and sensitive fractionation of plasma EVs with low variability, which will facilitate biomarker studies in large clinical cohorts.

Therapeutic Apheresis Using a β2-Microglobulin Removal Column Reduces Circulating Tumor Cell Count.

An elevated serum β2-microglobulin (β2M) level is indicative of impaired glomerular filtration and prerenal diseases, such as malignant tumors, autoimmune disorders, and liver diseases. An elevated serum β2M level has been shown to promote metastasis via the induction of epithelial-mesenchymal transition (EMT) in cancer cells. However, the therapeutic potential of targeting β2M remains unclear. Here, we aimed to investigate the efficacy of Filtor, a small polymethyl methacrylate fiber-based β2M removal column, in reducing the β2M level and suppressing cancer cell-induced EMT and metastasis. We assessed the effects of Filtor on the changes in metastasis based on the number of circulating tumor cells (CTCs), which reflects the post-EMT cancer cell population. We performed therapeutic apheresis using Filtor on a male patient with sinonasal neuroendocrine carcinoma, a female patient with a history of colorectal cancer, and another female patient with a history of pancreatic ductal adenocarcinoma. Significantly low serum β2M levels and CTC counts were observed immediately and 4 weeks after treatment compared with those in the pretreatment phase. Moreover, the CTC count immediately after therapeutic intervention was markedly reduced, likely because Filtor had trapped CTCs directly. These findings suggest that therapeutic apheresis with Filtor can prevent cancer metastasis and recurrence by directly removing CTCs.

The Innate Rates of Increase in Two Common Stored Grain Insects under Different Grain Storage Conditions and Times.

The determination of innate rate of increase (r) values under different grain storage conditions is critical for insect population predictions. The r values for Cryptolestes ferrugineus (Stephens) and Tribolium castaneum (Herbst) were calculated by using a new suggested method (continuous time analysis) and data from the literature, and these calculated r values were compared to identify the r values and carrying capacities under real grain storage conditions and times. The insects were reared in small glass vials (0.3 kg wheat), small PVC columns (2 kg wheat), large PVC columns (14 kg wheat), and shallow containers (14 kg wheat or wheat + cracked wheat). The wheat or cracked wheat had 13.8 to 14.5% moisture contents at different constant temperatures (17.5 to 42.5 °C) and fluctuating temperatures. The r values at the beginning of the population were the highest. Before r became negative, it gradually decreased with increasing time. After the r value became negative, it sometimes increased to positive; however, the rebounded r was much less than the initial r and gradually tended to stabilize within an up-and-down range. This up-and-down r was related to the carrying capacity. The larger the grain bulk, the higher the innate rate was for both species. The r values associated with 14 kg of wheat could be used to predict the insect population dynamics in stored grain bins.

The potential for CO2 storage in residual oil zones in onshore Australian basins

Geoscience Australia and CSIRO have collaborated to investigate whether residual oil zones (ROZs), sometimes associated with conventional Australian hydrocarbon plays, could provide a carbon dioxide (CO2) storage resource and enhance the storage capacity of depleted fields. Our petrophysical study demonstrates that ROZs occur in Australia’s hydrocarbon-rich regions, particularly in the Cooper-Eromanga Basin. ROZs with more than 10% residual oil saturation are uncommon, likely due to small original oil columns and lower residual saturations retained in sandstone reservoirs than in classic, carbonate-hosted North American ROZs. Extensive, reservoir-quality rock is found below the deepest occurring conventional oil in many of the fields in the Eromanga Basin, potentially offering significant CO2 storage capacity. Multiphase compositional flow modelling was used to estimate the CO2 storage efficiency of typical Australian ROZs. We developed a novel modelling methodology that first captures oil migration events leading to the formation of ROZs. Modelling CO2 storage over a 20-year injection period demonstrates that CO2-oil interactions increase the density and viscosity of CO2, enhancing CO2 sweep efficiency and lateral flow, improving storage efficiency. The extent of these effects depends on the quantity and spatial distribution of residual oil in place and the miscibility of CO2 at reservoir conditions.

Convenient pre-separation method depends on negative charges suitable for glycan sequencing analysis.

For precise structural analysis of complex glycans, it is necessary to fractionate glycans based on acidity prior to liquid chromatography-mass spectrometry analysis. Conventional fractionation using high-performance liquid chromatography is time consuming and not high throughput. Therefore, we developed a rapid method for the fractionation using a small cartridge column. This enables rapid multi-sample processing for glycan sequencing.

Efficient separation of radium from natural thorium using a mesoporous silica-supported composite resin with sulfonic acid groups for the acquisition of targeted α-nuclides 212Pb

212Pb serves as one of the most important nuclides for targeted alpha therapy (TAT), with good potential in the treatment of malignant tumors. However, medical and clinical applications are seriously limited by its extremely short supply, which strongly depends on the acquisition of its parent nuclides such as 228Ra or 224Ra. To address this issue, we conducted a systematic investigation on the separation of trace radium from natural thorium using the ion exchange chromatography method, and a novel porous silica-based composite resin functionalized with sulfonic acid groups was prepared, characterized, and employed. Experimental results showed that the silica-supported resin was successfully prepared with the sulfonic acid groups, and possessed small particle size, mesoporous pore structure and large specific surface area. The resin exhibited high selectivity toward thorium compared with barium, and the separation factor reached 123 at low acidity and at low temperature. The resin demonstrated a fast adsorption speed and an excellent separation performance, and the adsorption mechanism was confirmed based on the ion exchange between the sodium carried and the target cations. On the basis, column experiments were further conducted and finally the separation of trace radium from thorium was realized using a small column filled with about 1.0 g resin. The chemical recovery of radium reached 97 %, and the decontamination factor for thorium exceeded 40,000. This work verifies the feasibility of directly extracting 228Ra and 224Ra from natural thorium using the sulfonic acid group functionalized materials in the dilute nitric acid solution, and presents an excellent material and systematic experimental data on the separation activities, rendering this study particularly significant.

Behavior of large SCFST column with different confinement effects under pseudo-static loading

In this study, tests of twenty-four square concrete filled steel tubular (SCFST) columns with different structural sizes by the pseudo-static method were conducted to investigate the influence of the confinement effects. In addition, the structural size and the confinement effect are respectively represented by column width and width-to-thickness ratio. The experimental results, including the failure mode, the lateral load-displacement curves and the indices of the seismic performance were recorded and analyzed. The mode of failure is typical compressive flexure pattern. As the structural size increases or the confinement effect decreases, the nominal flexure strength of SCFST column decreases. Compared of the nominal flexural strength of small column, the influence of confinement effect is more significant on that of large column. The displacement ductility coefficient, the mean energy dissipation factor and the relative stiffness decrease with the increasing column width. Then enhanced confinement effect has no obvious influence on the displacement ductility coefficient of the SCFST column. When the confinement effect increases, the energy dissipation capacity of the SCFST column increases. Finally, the modified calculated method considering size effect is established, and this method is applicable to predict the bending moment of the SCFST column with different confinement effects and structural sizes.

Monolithic stationary phases prepared via cyclic anhydride ring-opening polymerization as tunable platforms for chromatographic applications.

Polymer monolithic stationary phases were prepared based on a cyclic anhydride as a reactive and tunable platform via ring-opening post-polymerization using primary amines, octadecylamine and benzylamine. The characterization techniques indicated the insertion of the functional groups into the original monoliths and confirmed the amidation reactions. The post-polymerization modification also improved the monolith's thermal and mechanical stability and induced significant improvement in their surface area. The stationary phases were synthesized inside small dimension stainless-steel columns (2.1 mm i.d. × 50 mm length). The prepared columns before and after modifications have been tested for the separation of the alkylbenzene series and some polycyclic aromatic hydrocarbons (PAHs) as model compounds. In all cases, the chromatographic performance in terms of the height equivalent to a theoretical plate on the functionalized monoliths was remarkably improved when compared with that on the unmodified monolith, which was between 9.59-39.49 μm and 4.08-31.50 μm using monoliths modified with octadecylamine and benzylamine, respectively. Under the same chromatographic conditions, the functionalization of monoliths with octadecylamine provided more hydrophobic interactions and enhanced the retention of alkylbenzenes, while the modification of monoliths with benzyl groups improved the separation and the retention of the PAHs through the strong π-π interactions. However, post-modification polymerization with octadecylamine and benzylamine enhanced the separation efficiency of the prepared columns toward all studied compounds. The repeatability of the injections on the same column and the reproducibility of the prepared columns have been studied for some selected parameters and estimated in terms of percent relative standard deviation (%RSD) for some of the studied compounds. The repeatability of the prepared columns was ≤9.42% (n = 5) based on run-to-run injections and ≤9.48% based on day-to-day injections for five successive days. The reproducibility levels, on the other hand, were ≤20.95% for all studied parameters in all cases. To assess their performance for the analysis of real samples, the applicability of the prepared columns was examined for the separation of the active ingredients extracted from some commercial pharmaceutical formulations and for the separation of tea water extract constituents. The validation data show the suitability of the columns for practical use in the routine analysis of these samples.

Relationship between diameter and bonding strength in resin/metal interface determined by small resin column shear test.

Relationship between diameter and bonding strength in resin/metal interface determined by small resin column shear test.

Design of a bus-based battery equalization with zero-current switching

A novel any cell to any cell equalization circuit topology with small column, low weight, low cost, and high efficiency is proposed for serial-connected batteries, especially suitable for unmanned aerial vehicles, satellites, and other fields with strict requirements on size, weight, reliability, and safety. To solve the problems of high switching losses in equalization circuits and low reliability of complex control schemes. A half-bridge circuit to convert the DC voltage of the battery into AC voltage is employed, and the energy is exchanged autonomously via the bus. At the same time, LC resonant circuit is used to achieve zero-current switching of the MOSFET, thus reduces greatly the switching losses. The working principle is analyzed by theoretical derivation, and the parameters of the circuit are optimized through circuit simulation. Furthermore, experiments which support six cells equalization are carried out to verify the feasibility and advantage of this topology. This equalization uses a bus-type architecture to improve equalization efficiency and topological flexibility making it even more valuable in the industrial and aerospace fields.

Performance of acid- and base-modified biochars for the removal of antibiotics from water under dynamic conditions

The increased recycling of treated municipal wastewater (TMW) to agricultural land is potentially hampered by the presence of antibiotic compounds, which may lead to the spread of antibiotic resistance. Biochar-based adsorption has been widely assessed as a means of polishing TMW to remove antibiotics prior to use; however, relatively little is known about how acid and base modification of biochar impacts its antibiotic retention capabilities, especially under the dynamic (flow-through) conditions inherent in proposed polishing systems. Using small columns of sand mixed with dairy manure or rice husk biochars (initial pH = 8.48 and 8.91, respectively) modified with acid or base, the retention of trimethoprim (TMP), sulfamethoxazole (SMZ), and sulfapyridine (SPD) was assessed. For SMZ and SPD, retention by the columns was consistently markedly lower than that measured in batch studies, indicating that equilibrium data may overestimate the retention of more realistic (dynamic) systems. TMP was completely retained by all biochars under both conditions. In columns, SMZ retention was strongly controlled by its pH-dependent speciation (greater retention at low pH, i.e., in acid modified biochars). SPD retention was strongly controlled by biochar surface area, which was markedly increased in the acid-modified dairy manure biochar and base-modified rice husk biochar. The findings indicate that acid or base modification of certain biochars may improve retention of sulfonamide antibiotics (which are known to be particularly difficult to remove from TMW) even under dynamic conditions. Biochar-based polishing systems comprising such biochars may offer potential in mitigating the spread of antibiotics, and hence antibiotic resistance, in agricultural settings.

Rescaling invariance and anomalous energy transport in a small vertical column of grains.

It is well known that energy dissipation and finite size can deeply affect the dynamics of granular matter, often making usual hydrodynamic approaches problematic. Here we report on the experimental investigation of a small model system, made of ten beads constrained into a 1D geometry by a narrow vertical pipe and shaken at the base by a piston excited by a periodic wave. Recording the beads motion with a high frame rate camera allows to investigate in detail the microscopic dynamics and test hydrodynamic and kinetic models. Varying the energy, we explore different regimes from fully fluidized to the edge of condensation, observing good hydrodynamic behavior down to the edge of fluidization, despite the small system size. Density and temperature fields for different system energies can be collapsed by suitable space and time rescaling, and the expected constitutive equationholds very well when the particle diameter is considered. At the same time, the balance between dissipated and fed energy is not well described by commonly adopted dependence due to the up-down symmetry breaking. Our observations, supported by the measured particle velocity distributions, show a different phenomenological temperature dependence, which yields equationsolutions in agreement with experimental results.

Improved sample introduction approach in hydrophilic interaction liquid chromatography to avoid breakthrough of proteins

When therapeutic proteins are analysed under hydrophilic interaction liquid chromatography (HILIC) conditions, there is an inherent mismatch between the sample diluent (proteins must be solubilised in aqueous media) and the mobile phase, which is mostly composed of aprotic solvent (acetonitrile). This difference in eluent strength between sample diluent and mobile phase is responsible for severe analyte breakthrough and peak distortion. As demonstrated with therapeutic proteins of different sizes (insulin of 6 kDa, anakinra of 17 kDa and rituximab subunits of 25 and 50 kDa), only very small volumes of 0.1–0.2 µL can be injected without breakthrough effects, when performing rapid analysis on short HILIC columns of 20–50 mm, leading to poor sensitivity. In order to avoid the undesired effect of the strong sample diluent, a special injection program should be preferred. This consists in the addition and automatic injection of a defined volume of weak solvent (acetonitrile) along with the sample to increase retention factors during sample loading. Various injection programs were tested, including the addition of a pre-injection or post-injection or both (bracketed injection) of acetonitrile plugs. Several weak to strong injection solvent ratios of 1:1, 1:2, 1:4 and 1:10 were tested. Our work proves that the addition of a pre-plug solvent with a weak vs. strong injection solvent ratio of 1:10 is a valuable strategy to inject relatively large volumes of proteins in HILIC, regardless of column dimensions, thus maximising sensitivity. No peak deformation or breakthrough was observed under these conditions. However, it is important to note that peak broadening (40 % larger peaks) was observed when the injection program increased the injection solvent ratio from 1:1 to 1:10. Finally, this strategy was applied to a wide range of therapeutic mAb products with different physico-chemical properties. In all cases, relatively large volumes can be successfully injected onto small volume HILIC columns using a purely aqueous sample diluent, as long as an appropriate (weak) solvent pre-injection is applied.